There are known in the art methods of nucleic acid sequencing based on successive cycles of incorporation of fluorescently labelled nucleic acid analogues. In such “sequencing by synthesis” or “cycle sequencing” methods the identity of the added base is determined after each nucleotide addition by detecting the fluorescent label.
In particular, U.S. Pat. No. 5,302,509 describes a method for sequencing a polynucleotide template which involves performing multiple extension reactions using a DNA polymerase or DNA ligase to successively incorporate labelled polynucleotides complementary to a template strand. In such a “sequencing by synthesis” reaction a new polynucleotide strand based-paired to the template strand is built up in the 5′ to 3′ direction by successive incorporation of individual nucleotides complementary to the template strand. The substrate nucleoside triphosphates used in the sequencing reaction are labelled at the 3′ position with different 3′ labels, permitting determination of the identity of the incorporated nucleotide as successive nucleotides are added.
The present inventors have developed methods of sequencing multiple nucleic acid molecules in parallel based on the use of arrays, wherein multiple template molecules immobilised on the array are sequenced in parallel. Such arrays may be single molecule arrays or clustered arrays.
The inventors have observed that when performing sequencing-by-synthesis using fluorescently labelled nucleotide analogues the brightness of the incorporated fluorophore diminishes at each cycle of nucleotide addition. This is a particular problem when sequencing nucleic acid templates on arrays, and more particularly arrays comprised of clusters of surface-bound DNA. Cycles of sequencing may stop at around cycle 8-10 due to loss of signal from the fluorescently labelled nucleotide analogue incorporated into extended strand complementary to the template, making it difficult to score accurately the identity of the incorporated base. Moving to a new area of the array that has not been previously scanned however clearly shows that the correct base can be accurately read. This is indicative of light-induced damage to the nucleic acid templates upon repeated exposures to the intense illumination used to read the incorporated fluorophores.
It is known in the art to add chemical antioxidants such as ascorbic acid (vitamin C) to fluorescent imaging buffers. It has, however, never previously been suggested to add such antioxidants to buffers used for detection/imaging of fluorophores incorporated in or attached to nucleic acid. More particularly, it has never previously been suggested to use antioxidants such as ascorbate as additives in buffers used for imaging of arrays during cycles of nucleic acid sequencing.
The inventors have now observed that the presence of one or more antioxidants in buffers used in any molecular biology technique requiring detection of a fluorescent moiety incorporated in or attached to a nucleic acid, which detection includes repeated or prolonged exposure to intense illumination, is surprisingly advantageous.